Our long term goal is to identify the molecular mechanisms underlying the proposed linkage between oxidative stress and the age-dependent loss of calcium regulation. Based on our previous findings that during aging multiple methionines in the calcium regulatory protein calmodulin (CaM) are oxidatively modified to their corresponding sulfoxides and the key role that CaM plays in intracellular signaling, we hypothesize that age- related decreases in CaM function play a major role in the loss of calcium homeostasis observed in senescent cells. The accumulation of oxidatively modified and functionally inactive CaM during aging is consistent with a decreased function of cellular repair and degradative enzymes in senescent animals. Thus the specific activity of methionine sulfoxide reductase (MsrA), which is able to repair oxidized CaM in vitro and restore the ability of oxidized CaM to activate the plasma membrane Ca- ATPase (PMCA), may be compromised during aging. Likewise, age- related decreases in the function of the proteasome, which normally degrades oxidized proteins, may result in the accumulation of inactive CaM. Therefore, to identify the cellular effects of CaM oxidation, we propose to test whether changes in CaM function observed in vitro using purified CaM are important in vivo for modulating cell function. These measurements will combine biochemical measurements of protein function using genetically engineered CaM mutants with altered sensitivities to oxidative stress with well defined measurements of cellular function and calcium regulation of neurons in cell culture. It is our expectation that the oxidative modification of CaM correlates with the loss of calcium regulation and enhanced sensitivity of neurons to oxidative stress. We therefore propose the following specific aims: (1) Identify how methionine oxidation in CaM alters target protein activation, (2) Investigate the cellular consequences of CaM oxidation on calcium homeostasis and neuronal visibility, (3) Determine the physiological role of MsrA in the maintenance of cell function, and (4) Investigate functional modifications to cellular repair and degradative systems during aging. An understanding of cellular mechanisms that modify calcium homeostasis under conditions of oxidative stress in the role of CaM oxidation in modifying cellular survival will be important to the development of possible therapies that could alleviate the decline in cellular functions associated with aging.